Generation of plants with altered oil, protein, or fiber content

ABSTRACT

The present invention is directed to plants that display an improved oil quantity phenotype or an improved meal quality phenotype due to altered expression of an HIO nucleic acid. The invention is further directed to methods of generating plants with an improved oil quantity phenotype or improved meal quality phenotype.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application No.60/870,355, filed Dec. 15, 2006, the entirety of which is incorporatedherein by reference.

FIELD OF THE DISCLOSURE

The present disclosure is related to modified plants with altered oil,protein, and/or fiber content, as well as methods of making modifiedplants having altered oil, protein, and/or fiber content and producingoil from such plants.

BACKGROUND

The ability to manipulate the composition of crop seeds, particularlythe content and composition of seed oil and protein, as well as theavailable metabolizable energy (“AME”) in the seed meal in livestock,has important applications in the agricultural industries, relating bothto processed food oils and to animal feeds. Seeds of agricultural cropscontain a variety of valuable constituents, including oil, protein andstarch. Industrial processing can separate some or all of theseconstituents for individual sale in specific applications. For instance,nearly 60% of the U.S. soybean crop is crushed by the soy processingindustry. Soy processing yields purified oil, which is sold at highvalue, while the remaining seed meal is sold for livestock feed (U.S.Soybean Board, 2001 Soy Stats). Canola seed is also crushed to produceoil and the co-product canola meal (Canola Council of Canada). Canolameal contains a high percentage of protein and a good balance of aminoacids but because it has a high fiber and phytate content, it is notreadily digested by livestock (Slominski, B. A., et al., 1999Proceedings of the 10^(th) International Rapeseed Congress, Can berra,Australia) and has a lower value than soybean meal.

Over 55% of the corn produced in the U.S. is used as animal feed (IowaCorn Growers Association). The value of the corn is directly related toits ability to be digested by livestock. Thus, it is desirable tomaximize both oil content of seeds and the AME of meal. For processedoilseeds such as soy and canola, increasing the absolute oil content ofthe seed will increase the value of such grains, while increasing theAME of meal will increase its value. For processed corn, either anincrease or a decrease in oil content may be desired, depending on howthe other major constituents are to be used. Decreasing oil may improvethe quality of isolated starch by reducing undesired flavors associatedwith oil oxidation. Alternatively, when the starch is used for ethanolproduction, where flavor is unimportant, increasing oil content mayincrease overall value.

In many feed grains, such as corn and wheat, it is desirable to increaseseed oil content, because oil has higher energy content than other seedconstituents such as carbohydrate. Oilseed processing, like most grainprocessing businesses, is a capital-intensive business; thus smallshifts in the distribution of products from the low valued components tothe high value oil component can have substantial economic impacts forgrain processors. In addition, increasing the AME of meal by adjustingseed protein and fiber content and composition, without decreasing seedoil content, can increase the value of animal feed.

Biotechnological manipulation of oils has been shown to providecompositional alteration and improvement of oil yield. Compositionalalterations include high oleic acid soybean and corn oil (U.S. Pat. Nos.6,229,033 and 6,248,939), and laurate-containing seeds (U.S. Pat. No.5,639,790), among others. Work in compositional alteration haspredominantly focused on processed oilseeds, but has been readilyextendable to non-oilseed crops, including corn. While there isconsiderable interest in increasing oil content, the only currentlypracticed biotechnology in this area is High-Oil Corn (HOC) technology(DuPont, U.S. Pat. No. 5,704,160). HOC employs high oil pollinatorsdeveloped by classical selection breeding along with elite(male-sterile) hybrid females in a production system referred to asTopCross. The TopCross High Oil system raises harvested grain oilcontent in maize from about 3.5% to about 7%, improving the energycontent of the grain.

While it has been fruitful, the HOC production system has inherentlimitations. First, the system of having a low percentage of pollinatorsresponsible for an entire field's seed set contains inherent risks,particularly in drought years. Second, oil content in current HOC fieldshas not been able to achieve seed oil content above 9%. Finally,high-oil corn is not primarily a biochemical change, but rather ananatomical mutant (increased embryo size) that has the indirect resultof increasing oil content. For these reasons, an alternative high oilstrategy, particularly one that derives from an altered biochemicaloutput, would be especially valuable.

Manipulation of seed composition has identified several components thatimprove the nutritive quality, digestibility, and AME in seed meal.Increasing the lysine content in canola and soybean (Falco et al., 1995Bio/Technology 13:577-582) increases the availability of this essentialamino acid and decreases the need for nutritional supplements. Soybeanvarieties with increased seed protein were shown to contain considerablymore metabolizable energy than conventional varieties (Edwards et al.,1999, Poultry Sci. 79:525-527). Decreasing the phytate content of cornseed has been shown to increase the bioavailability of amino acids inanimal feeds (Douglas et al., 2000, Poultry Sci. 79:1586-1591) anddecreasing oligosaccharide content in soybean meal increases themetabolizable energy in the meal (Parsons et al., 2000, Poultry Sci.79:1127-1131).

Soybean and canola are the most obvious target crops for the processedoil and seed meal markets since both crops are crushed for oil and theremaining meal sold for animal feed. A large body of commercial work(e.g., U.S. Pat. No. 5,952,544; PCT Application No. WO9411516)demonstrates that Arabidopsis is an excellent model for oil metabolismin these crops. Biochemical screens of seed oil composition haveidentified Arabidopsis genes for many critical biosynthetic enzymes andhave led to identification of agronomically important gene orthologs.For instance, screens using chemically mutagenized populations haveidentified lipid mutants whose seeds display altered fatty acidcomposition (Lemieux et al., 1990, Theor. Appl. Genet. 80: 234-240;James and Dooner, 1990, Theor. Appl. Genet. 80: 241-245). T-DNAmutagenesis screens (Feldmann et al., 1989, Science 243: 1351-1354) thatdetected altered fatty acid composition identified the omega 3desaturase (FAD3) and delta-12 desaturase (FAD2) genes (U.S. Pat. No.5,952,544; Yadav et al., 1993, Plant Physiol. 103: 467-476; Okuley etal., 1994, Plant Cell 6(1):147-158). A screen which focused on oilcontent rather than oil quality, analyzed chemically-induced mutants forwrinkled seeds or altered seed density, from which altered seed oilcontent was inferred (Focks and Benning, 1998, Plant Physiol.118:91-101).

Another screen, designed to identify enzymes involved in production ofvery long chain fatty acids, identified a mutation in the gene encodinga diacylglycerol acyltransferase (DGAT) as being responsible for reducedtriacyl glycerol accumulation in seeds (Katavic V et al., 1995, PlantPhysiol. 108(1):399-409). It was further shown that seed-specificover-expression of the DGAT cDNA was associated with increased seed oilcontent (Jako et al., 2001, Plant Physiol. 126(2):861-74). Arabidopsisis also a model for understanding the accumulation of seed componentsthat affect meal quality. For example, Arabidopsis contains albumin andglobulin seed storage proteins found in many dicotyledonous plantsincluding canola and soybean (Shewry 1995, Plant Cell 7:945-956). Thebiochemical pathways for synthesizing components of fiber, such ascellulose and lignin, are conserved within the vascular plants, andmutants of Arabidopsis affecting these components have been isolated(reviewed in Chapel and Carpita 1998, Current Opinion in Plant Biology1:179-185).

Activation tagging in plants refers to a method of generating randommutations by insertion of a heterologous nucleic acid constructcomprising regulatory sequences (e.g., an enhancer) into a plant genome.The regulatory sequences can act to enhance transcription of one or morenative plant genes; accordingly, activation tagging is a fruitful methodfor generating gain-of-function, generally dominant mutants (see, e.g.,Hayashi et al., 1992, Science 258: 1350-1353; Weigel D et al., 2000,Plant Physiology, 122:1003-1013). The inserted construct provides amolecular tag for rapid identification of the native plant whosemis-expression causes the mutant phenotype. Activation tagging may alsocause loss-of-function phenotypes. The insertion may result indisruption of a native plant gene, in which case the phenotype isgenerally recessive.

Activation tagging has been used in various species, including tobaccoand Arabidopsis, to identify many different kinds of mutant phenotypesand the genes associated with these phenotypes (Wilson et al., 1996,Plant Cell 8: 659-671; Schaffer et al., 1998, Cell 93: 1219-1229;Fridborg et al., 1999, Plant Cell 11: 1019-1032; Kardailsky et al.,1999, Science 286: 1962-1965; and Christensen S et al., 1998, 9^(th)International Conference on Arabidopsis Research, Univ. ofWisconsin-Madison, June 24-28, Abstract 165).

SUMMARY

Provided herein are modified plants having an altered phenotype.Modified plants with an altered phenotype may include an improved oilquantity and/or an improved meal quality phenotype. The alteredphenotype in a modified plant may also include altered oil, protein,and/or fiber content in any part of the modified plant, for example inthe seeds. In some embodiments of a modified plant, the alteredphenotype is an increase in the oil content of the seed (a high oilphenotype). In other embodiments, the altered phenotype may be anincrease in protein content in the seed and/or a decrease in the fibercontent of the seed. Also provided is seed meal derived from the seedsof modified plants, wherein the seeds have altered protein contentand/or altered fiber content. Further provided is oil derived from theseeds of modified plants, wherein the seeds have altered oil content.Any of these changes can lead to an increase in the AME from the seed orseed meal from modified plants, relative to control or wild-type plants.Also provided herein is meal, feed, or food produced from any part ofthe modified plant with an altered phenotype.

In certain embodiments, the disclosed modified plants include transgenicplants having a transformation vector comprising a HIO nucleotidesequence (or HIO gene alias) that encodes or is complementary to asequence that encodes a “HIO” polypeptide. In particular embodiments,expression of a HIO polypeptide in a transgenic plant causes an alteredoil content, an altered protein content, and/or an altered fiber contentin the transgenic plant. In preferred embodiments, the transgenic plantis selected from the group consisting of plants of the Brassica species,including canola and rapeseed, soy, corn, sunflower, cotton, cocoa,safflower, oil palm, coconut palm, flax, castor, peanut, wheat, oat andrice. Also provided is a method of producing oil or seed meal,comprising growing the transgenic plant and recovering oil and/or seedmeal from said plant. The disclosure further provides feed, meal, grain,or seed comprising a nucleic acid sequence that encodes a HIOpolypeptide. The disclosure also provides feed, meal, grain, or seedcomprising the HIO polypeptide, or an ortholog or paralog thereof.

Examples of the disclosed transgenic plant are produced by a method thatcomprises introducing into progenitor cells of the plant a planttransformation vector comprising a HIO nucleotide sequence that encodes,or is complementary to a sequence that encodes, a HIO polypeptide, andgrowing the transformed progenitor cells to produce a transgenic plant,wherein the HIO polynucleotide sequence is expressed, causing an alteredphenotype in the transgenic plant. In some specific, non-limitingexamples, the method produces transgenic plants wherein expression ofthe HIO polypeptide causes a high (increased) oil, high (increased)protein, and/or low (decreased) fiber phenotype in the transgenic plant,relative to control, non-transgenic, or wild-type plants.

Additional methods are disclosed herein of generating a plant having analtered phenotype, wherein a plant is identified that has a mutation oran allele in its HIO nucleic acid sequence that results in an alteredphenotype, compared to plants lacking the mutation or allele. Themutated plant can be generated using one or more mutagens, for example achemical mutagen, radiation, or ultraviolet light. In some embodimentsof the method, the plant is bred to generate progeny which inherit theallele and express the altered phenotype. In particular embodiments ofthe method, the method employs candidate gene/QTL methodology or TILLINGmethodology.

Also provided herein is a modified plant cell having an alteredphenotype. In some embodiments, the modified plant cell includes atransformation vector comprising a HIO nucleotide sequence that encodesor is complementary to a sequence that encodes a HIO polypeptide. Inpreferred embodiments, the transgenic plant cell is selected from thegroup consisting of plants of the Brassica species, including canola andrapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oil palm,coconut palm, flax, castor, peanut, wheat, oat and rice. In otherembodiments, the plant cell is a seed, pollen, propagule, or embryocell. The disclosure also provides plant cells from a plant that is thedirect progeny or the indirect progeny of a plant grown from saidprogenitor cells.

DETAILED DESCRIPTION Terms

Unless otherwise indicated, all technical and scientific terms usedherein have the same meaning as they would to one skilled in the art ofthe present disclosure. Practitioners are particularly directed toSambrook et al. (Molecular Cloning: A Laboratory Manual (SecondEdition), Cold Spring Harbor Press, Plainview, N.Y., 1989) and Ausubel FM et al. (Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 1993) for definitions and terms of the art. It is to beunderstood that this disclosure is not limited to the particularmethodology, protocols, and reagents described, as these may vary.

As used herein, the term “altered phenotype” refers to plants, or anypart of a plant (for example, seeds, or meal produced from seeds), withan altered oil, protein, and/or fiber content (phenotype). As providedherein, altered oil, protein (for example, digestible protein) and/orfiber content includes either an increased or decreased level of oil,protein (for example, digestible protein) and/or fiber content inplants, seeds or seed meal. Any combination of these changes can lead toan altered phenotype. For example, in one specific non-limiting example,an altered phenotype can refer to increased oil and decreased fibercontent. In another specific non-limiting example, an altered phenotypecan refer to unchanged protein and decreased fiber content. In anotherspecific non-limiting example, an altered phenotype can refer toincreased oil and protein and decreased fiber. In yet other non-limitingexamples, an altered phenotype can refer to increased oil and proteinand unchanged fiber content; unchanged oil, increased protein, anddecreased fiber content; or increased oil, increased protein, anddecreased fiber content. It is also provided that any combination ofthese changes can lead to an increase in the AME (availablemetabolizable energy) from the seed or meal generated from the seed. Analtered phenotype also includes an improved seed quality (ISQ) phenotypeor an improved seed meal quality phenotype.

As used herein, the term “available metabolizable energy” (AME) refersto the amount of energy in the feed that is able to be extracted bydigestion in an animal and is correlated with the amount of digestibleprotein and oil available in animal meal. AME is determined byestimating the amount of energy in the feed prior to feeding andmeasuring the amount of energy in the excreta of the animal followingconsumption of the feed. In one specific, non-limiting example, amodified plant with an increase in AME includes modified plants withaltered seed oil, digestible protein, total protein and/or fibercontent, resulting in an increase in the value of animal feed derivedfrom the seed.

As used herein, the term “content” refers to the type and relativeamount of, for instance, a seed or seed meal component.

As used herein, the term “seed oil” refers to the total amount of oilwithin the seed.

As used herein, the term “seed fiber” refers to non-digestiblecomponents of the plant seed including cellular components such ascellulose, hemicellulose, pectin, lignin, and phenolics.

As used herein, the term “meal” refers to seed components remainingfollowing the extraction of oil from the seed. Examples of components ofmeal include protein and fiber.

As used herein, the term “seed total protein” refers to the total amountof protein within the seed.

As used herein, the term “seed digestible protein” refers to the seedprotein that is able to be digested by enzymes in the digestive track ofan animal. It is a subset of the total protein content.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

A “heterologous” nucleic acid construct or sequence has a portion of thesequence that is not native to the plant cell in which it is expressed.Heterologous, with respect to a control sequence refers to a controlsequence (i.e. promoter or enhancer) that does not function in nature toregulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell by infection, transfection,microinjection, electroporation, or the like. A “heterologous” nucleicacid construct may contain a control sequence/DNA coding sequencecombination that is the same as, or different from, a controlsequence/DNA coding sequence combination found in the native plant.Specific, non-limiting examples of a heterologous nucleic acid sequenceinclude a HIO nucleic acid sequence, or a fragment, derivative(variant), or ortholog or paralog thereof.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, which may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons) and non-transcribed regulatory sequences.

The term “homolog” refers to any gene that is related to a referencegene by descent from a common ancestral DNA sequence. The term“ortholog” refers to homologs in different species that evolved from acommon ancestral gene by speciation. Typically, orthologs retain thesame or similar function despite differences in their primary structure(mutations). The term “paralog” refers to homologs in the same speciesthat evolved by genetic duplication of a common ancestral gene. In manycases, paralogs exhibit related (but not always identical functions). Asused herein, the term homolog encompasses both orthologs and paralogs.To the extent that a particular species has evolved multiple relatedgenes from an ancestral DNA sequence shared with another species, theterm ortholog can encompass the term paralog.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all as a result of deliberate humanintervention.

As used herein, the term “gene expression” refers to the process bywhich a polypeptide is produced based on the nucleic acid sequence of agene. The process includes both transcription and translation;accordingly, “expression” may refer to either a polynucleotide orpolypeptide sequence, or both. Sometimes, expression of a polynucleotidesequence will not lead to protein translation. “Over-expression” refersto increased expression of a polynucleotide and/or polypeptide sequencerelative to its expression in a wild-type (or other reference [e.g.,non-transgenic]) plant and may relate to a naturally-occurring ornon-naturally occurring sequence. “Ectopic expression” refers toexpression at a time, place, and/or increased level that does notnaturally occur in the non-modified or wild-type plant.“Under-expression” refers to decreased expression of a polynucleotideand/or polypeptide sequence, generally of an endogenous gene, relativeto its expression in a wild-type plant. The terms “mis-expression” and“altered expression” encompass over-expression, under-expression, andectopic expression.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, includes “transfection,” “transformation,” and“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

As used herein, a “plant cell” refers to any cell derived from a plant,including cells from undifferentiated tissue (e.g., callus), as well asfrom plant seeds, pollen, propagules, and embryos.

As used herein, the terms “native” and “wild-type” relative to a givenplant trait or phenotype refers to the form in which that trait orphenotype is found in the same variety of plant in nature. In oneembodiment, a wild-type or native plant is also a control plant. Inanother embodiment, a wild-type or native plant is a non-transgenic ornon-mutated plant. In yet another embodiment, a wild-type or nativeplant is a non-modified plant.

As used herein, the term “modified” regarding a plant, refers to a plantwith an altered phenotype (for example, a plant generated by geneticengineering, mutagenesis, or breeding methods). A genetically engineeredplant can also be a transgenic plant. In particular embodiments,modified plants generated by breeding methods are first mutagenizedusing any one of a variety of mutagens, such as a chemical mutagen,radiation, or ultraviolet light. Modified plants can have anycombination of an altered oil content, an altered protein content,and/or an altered fiber content in any part of the transgenic plant, forexample the seeds, relative to a similar non-modified plant.

As used herein, the term “altered” refers to a change (either anincrease or a decrease) of a plant trait or phenotype (for example, oilcontent, protein content, and/or fiber content) in a modified plant,relative to a similar non-modified plant. In one specific, non-limitingexample, a modified plant with an altered trait includes a plant with anincreased oil content, increased protein content, and/or decreased fibercontent relative to a similar non-modified plant. In another specific,non-limiting example, a modified plant with an altered trait includesunchanged oil content, increased protein content, and/or decreased fibercontent relative to a similar non-modified plant. In yet anotherspecific, non-limiting example, a modified plant with an altered traitincludes an increased oil content, increased protein content, and/orunchanged fiber content relative to a similar non-modified plant.

An “interesting phenotype (trait)” with reference to a modified plantrefers to an observable or measurable phenotype demonstrated by a T1and/or subsequent generation plant, which is not displayed by thecorresponding non-modified plant (i.e., a genotypically similar plantthat has been raised or assayed under similar conditions). Aninteresting phenotype may represent an improvement in the plant (forexample, increased oil content, increased protein content, and/ordecreased fiber content in seeds of the plant) or may provide a means toproduce improvements in other plants. An “improvement” is a feature thatmay enhance the utility of a plant species or variety by providing theplant with a unique and/or novel phenotype or quality. Such modifiedplants may have an improved phenotype, such as an altered oil, protein,and/or fiber phenotype. Meal generated from seeds of a modified plantwith an improved phenotype can have improved (increased) meal quality.In a specific, non-limiting example of meal with an improved (increased)quality phenotype, meal is generated from a seed of a modified plant,wherein the seed has increased protein content and/or decreased fibercontent, relative to a similar non-modified plant.

The phrase “altered oil content phenotype” refers to a measurablephenotype of a modified plant, where the plant displays a statisticallysignificant increase or decrease in overall oil content (i.e., thepercentage of seed mass that is oil), as compared to the similar, butnon-modified (for example, a non-transgenic or a non-mutated) plant. Ahigh oil phenotype refers to an increase in overall oil content. Anincrease in oil content includes, in various embodiments, about a 1.0%,1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 7.5%, 10%, or moreincrease in oil content. Likewise, a decrease in oil content includesabout a 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 7.5%, 10%,or more decrease in oil content, in various embodiments.

The phrase “altered protein content phenotype” refers to measurablephenotype of a modified plant, where the plant displays a statisticallysignificant increase or decrease in overall protein content (i.e., thepercentage of seed mass that is protein), as compared to the similar,but non-modified (for example, non-transgenic or non-mutated) plant. Ahigh protein phenotype refers to an increase in overall protein content.An increase in protein content includes, in various embodiments, about a1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 7.5%, 10%, or moreincrease in total protein content. Likewise, an increase in digestibleprotein content includes, in various embodiments, about a 1.0%, 1.5%,2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 7.5%, 10%, or more increase indigestible protein content. A decrease in protein content includes abouta 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 7.5%, 10%, ormore decrease in total protein content, in various embodiments.Likewise, a decrease in digestible protein content includes, in variousembodiments, about a 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%,5.0%, 7.5%, 10%, or more decrease in digestible protein content. Thephrase “altered fiber content phenotype” refers to measurable phenotypeof a modified plant, where the plant displays a statisticallysignificant increase or decrease in overall fiber content (i.e., thepercentage of seed mass that is fiber), as compared to the similar, butnon-modified (for example, non-transgenic or non-mutated) plant. A lowfiber phenotype refers to decrease in overall fiber content. An increasein fiber content includes about a 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%,4.0%, 4.5%, 5.0%, 7.5%, 10%, or more increase in fiber content.Likewise, a decrease in fiber content includes about a 1.0%, 1.5%, 2.0%,2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 7.5%, 10%, or more decrease in fibercontent.

As used herein, a “mutant” or “mutated” polynucleotide sequence or genediffers from the corresponding wild-type polynucleotide sequence or geneeither in terms of sequence or expression, where the differencecontributes to an altered plant phenotype or trait. Relative to a plantor plant line, the term “mutant” or “mutated” refers to a plant or plantline which has an altered plant phenotype or trait, where the alteredphenotype or trait is associated with the altered expression of awild-type polynucleotide sequence or gene. The mutated polynucleotidesequence or gene can be generated by genetic engineering methods (suchas activation tagging or transformation), by using one or more mutagens(for example, chemical mutagens, radiation, or ultraviolet light), or byusing methods to alter a DNA sequence (for example, error prone PCR, DNAshuffling molecular breeding, site-directed mutagenesis, or introducingthe gene into a mutagenizing organism such as E. coli or yeast strainsthat are deficient in DNA repair activity).

As used herein, the term “T1” refers to the generation of plants fromthe seed of T0 plants. The T1 generation is the first set of modifiedplants that can be selected by application of a selection agent, e.g.,an antibiotic or herbicide, for which the modified plant contains thecorresponding resistance gene. The term “T2” refers to the generation ofplants by self-fertilization of the flowers of T1 plants, previouslyselected as being modified. T3 plants are generated from T2 plants, etc.As used herein, the “direct progeny” of a given plant derives from theseed (or, sometimes, other tissue) of that plant and is in theimmediately subsequent generation; for instance, for a given lineage, aT2 plant is the direct progeny of a T1 plant. The “indirect progeny” ofa given plant derives from the seed (or other tissue) of the directprogeny of that plant, or from the seed (or other tissue) of subsequentgenerations in that lineage; for instance, a T3 plant is the indirectprogeny of a T1 plant.

As used herein, the term “plant part” includes any plant organ ortissue, including, without limitation, seeds, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores. Plant cells can be obtained fromany plant organ or tissue and cultures prepared therefrom. Providedherein is a modified plant cell having an altered phenotype. Inparticular embodiments, the modified plant cell is a transgenic plantcell. The transgenic plant cell includes a transformation vectorcomprising an HIO nucleotide sequence that encodes or is complementaryto a sequence that encodes an HIO polypeptide. In preferred embodiments,the transgenic plant cell is selected from the group consisting ofplants of the Brassica species, including canola and rapeseed, soy,corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax,castor, peanut, wheat, oat and rice. In other embodiments, the plantcell is a seed, pollen, propagule, or embryo cell. The disclosure alsoprovides plant cells from a plant that is the direct progeny or theindirect progeny of a plant grown from said progenitor cells. The classof plants which can be used in the methods of the present invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledonous anddicotyledonous plants.

As used herein, “transgenic plant” includes a plant that compriseswithin its genome a heterologous polynucleotide. The heterologouspolynucleotide can be either stably integrated into the genome, or canbe extra-chromosomal. Preferably, the polynucleotide of the presentinvention is stably integrated into the genome such that thepolynucleotide is passed on to successive generations. A plant cell,tissue, organ, or plant into which the heterologous polynucleotides havebeen introduced is considered “transformed,” “transfected,” or“transgenic.” Direct and indirect progeny of transformed plants or plantcells that also contain the heterologous polynucleotide are alsoconsidered transgenic.

Disclosed herein are modified plants having an altered phenotype.Modified plants with an altered phenotype may include an improved(increased) oil quantity and/or an improved (increased) meal quality, ascompared to the similar, but non-modified (for example, non-transgenicor non-mutated) plant. Modified plants with an altered phenotype mayinclude altered oil, protein, and/or fiber content in any part of themodified plant, for example in the seeds, as compared to the similar,but non-modified (for example, non-transgenic or non-mutated) plant. Insome embodiments of a modified plant, for example in plants with animproved or increased oil content phenotype, the altered phenotypeincludes an increase in the oil content of the seed (a high oilphenotype) from the plant, as compared to the similar, but non-modified(non-transgenic or non-mutated) plant. An increase in oil contentincludes, in various embodiments, about a 1.0%, 1.5%, 2.0%, 2.5%, 3.0%,3.5%, 4.0%, 4.5%, 5.0%, 7.5%, 10%, or more increase in oil content. Thealtered phenotype can be an increase in one or more fatty acids, such asoleic acid, with a concominant decrease in other fatty acids such aslinoleic or linolinic acids. A change in fatty acid content includesabout a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or moreincrease in a specific fatty acid. In other embodiments of a modifiedplant, for example in plants with an improved or increased meal qualityphenotype, the altered phenotype may be an increase in protein contentin the seed and/or a decrease in the fiber content of the seed. Anincrease in protein content includes about a 1.0%, 1.5%, 2.0%, 2.5%,3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 7.5%, 10%, or more increase in proteincontent, for instance total protein content or digestible proteincontent. This change in seed protein content can be the result ofaltered amounts of seed storage proteins such as albumins, globulinsprolamins, and glutelins. A decrease in fiber content includes about a1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 7.5%, 10%, or moredecrease in fiber content. This change in fiber content can be theresult of altered amounts of fibrous components such as cellulose,hemicellulose, lignin and pectins.

Also provided is seed meal derived from the seeds of modified plants,wherein the seeds have altered (for example, increased) protein (forexample, digestible) content and/or altered (for example, decreased)fiber content. Further provided is oil derived from the seeds ofmodified plants, wherein the seeds have altered oil content. Any ofthese changes can lead to an increase in the AME from the seed or seedmeal from modified plants, relative to control, non-transgenic, orwild-type plants. An increase in the AME includes about a 1.0%, 1.5%,2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 7.5%, 10%, or more increase inAME in the seed or seed meal, in various embodiments. Also providedherein is meal, feed, or food produced from any part of the modifiedplant with an altered phenotype.

In certain embodiments, the disclosed transgenic plants comprise atransformation vector comprising a HIO nucleotide sequence that encodesor is complementary to a sequence that encodes a “HIO” polypeptide. Inparticular embodiments, expression of a HIO polypeptide in a transgenicplant causes an altered oil content, an altered protein content, and/oran altered fiber content in the transgenic plant. In preferredembodiments, the transgenic plant is selected from the group consistingof plants of the Brassica species, including canola and rapeseed, soy,corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax,castor, peanut, wheat, oat and rice. Also provided is a method ofproducing oil or seed meal, comprising growing the transgenic plant andrecovering oil and/or seed meal from said plant. The disclosure furtherprovides feed, meal, grain, or seed comprising a nucleic acid sequencethat encodes a HIO polypeptide. The disclosure also provides feed, meal,grain, or seed comprising the HIO polypeptide, or an ortholog or paralogthereof.

Various methods for the introduction of a desired polynucleotidesequence encoding the desired protein into plant cells are available andknown to those of skill in the art and include, but are not limited to:(1) physical methods such as microinjection, electroporation, andmicroprojectile mediated delivery (biolistics or gene gun technology);(2) virus mediated delivery methods; and (3) Agrobacterium-mediatedtransformation methods.

The most commonly used methods for transformation of plant cells are theAgrobacterium-mediated DNA transfer process and the biolistics ormicroprojectile bombardment mediated process (i.e., the gene gun).Typically, nuclear transformation is desired but where it is desirableto specifically transform plastids, such as chloroplasts or amyloplasts,plant plastids may be transformed utilizing a microprojectile-mediateddelivery of the desired polynucleotide.

Agrobacterium-mediated transformation is achieved through the use of agenetically engineered soil bacterium belonging to the genusAgrobacterium. A number of wild-type and disarmed strains ofAgrobacterium tumefaciens and Agrobacterium rhizogenes harboring T1 orRi plasmids can be used for gene transfer into plants. Gene transfer isdone via the transfer of a specific DNA known as “T-DNA” that can begenetically engineered to carry any desired piece of DNA into many plantspecies.

Agrobacterium-mediated genetic transformation of plants involves severalsteps. The first step, in which the virulent Agrobacterium and plantcells are first brought into contact with each other, is generallycalled “inoculation.” Following the inoculation, the Agrobacterium andplant cells/tissues are permitted to be grown together for a period ofseveral hours to several days or more under conditions suitable forgrowth and T-DNA transfer. This step is termed “co-culture.” Followingco-culture and T-DNA delivery, the plant cells are treated withbactericidal or bacteriostatic agents to kill or limit the growth of theAgrobacterium remaining in contact with the explant and/or in the vesselcontaining the explant. If this is done in the absence of any selectiveagents to promote preferential growth of transgenic versusnon-transgenic plant cells, then this is typically referred to as the“delay” step. If done in the presence of selective pressure favoringtransgenic plant cells, then it is referred to as a “selection” step.When a “delay” is used, it is typically followed by one or more“selection” steps.

With respect to microprojectile bombardment (U.S. Pat. No. 5,550,318;U.S. Pat. No. 5,538,880, U.S. Pat. No. 5,610,042; and PCT Publication WO95/06128; each of which is specifically incorporated herein by referencein its entirety), particles are coated with nucleic acids and deliveredinto cells by a propelling force. Exemplary particles include thosecomprised of tungsten, platinum, and preferably, gold.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System(BioRad, Hercules, Calif.), which can be used to propel particles coatedwith DNA or cells through a screen, such as a stainless steel or Nytexscreen, onto a filter surface covered with monocot plant cells culturedin suspension.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. Examples of species thathave been transformed by microprojectile bombardment include monocotspecies such as maize (PCT Publication No. WO 95/06128), barley, wheat(U.S. Pat. No. 5,563,055, incorporated herein by reference in itsentirety), rice, oat, rye, sugarcane, and sorghum, as well as a numberof dicots including tobacco, soybean (U.S. Pat. No. 5,322,783,incorporated herein by reference in its entirety), sunflower, peanut,cotton, tomato, and legumes in general (U.S. Pat. No. 5,563,055,incorporated herein by reference in its entirety).

To select or score for transformed plant cells regardless oftransformation methodology, the DNA introduced into the cell contains agene that functions in a regenerable plant tissue to produce a compoundthat confers upon the plant tissue resistance to an otherwise toxiccompound. Genes of interest for use as a selectable, screenable, orscorable marker would include but are not limited to GUS, greenfluorescent protein (GFP), luciferase (LUX), antibiotic or herbicidetolerance genes. Examples of antibiotic resistance genes include thepenicillins, kanamycin, neomycin, G418, bleomycin, methotrexate (andtrimethoprim), chloramphenicol, and tetracycline. Polynucleotidemolecules encoding proteins involved in herbicide tolerance are known inthe art, and include, but are not limited to a polynucleotide moleculeencoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) describedin U.S. Pat. No. 5,627,061, U.S. Pat. No. 5,633,435, and U.S. Pat. No.6,040,497 and aroA described in U.S. Pat. No. 5,094,945 for glyphosatetolerance; a polynucleotide molecule encoding bromoxynil nitrilase (Bxn)described in U.S. Pat. No. 4,810,648 for Bromoxynil tolerance; apolynucleotide molecule encoding phytoene desaturase (crtl) described inMisawa et al., (Plant J. 4:833-840, 1993) and Misawa et al., (Plant J.6:481-489, 1994) for norflurazon tolerance; a polynucleotide moleculeencoding acetohydroxyacid synthase (AHAS, also known as ALS) describedin Sathasiivan et al. (Nucl. Acids Res. 18:2188-2193, 1990) fortolerance to sulfonylurea herbicides; and the bar gene described inDeBlock, et al., (EMBO J. 6:2513-2519, 1987) for glufosinate andbialaphos tolerance.

The regeneration, development, and cultivation of plants from varioustransformed explants are well documented in the art. This regenerationand growth process typically includes the steps of selecting transformedcells and culturing those individualized cells through the usual stagesof embryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil. Cells that survive the exposure to the selectiveagent, or cells that have been scored positive in a screening assay, maybe cultured in media that supports regeneration of plants. Developingplantlets are transferred to soil less plant growth mix, and hardenedoff, prior to transfer to a greenhouse or growth chamber for maturation.

The present invention can be used with any transformable cell or tissue.By transformable as used herein is meant a cell or tissue that iscapable of further propagation to give rise to a plant. Those of skillin the art recognize that a number of plant cells or tissues aretransformable in which after insertion of exogenous DNA and appropriateculture conditions the plant cells or tissues can form into adifferentiated plant. Tissue suitable for these purposes can include butis not limited to immature embryos, scutellar tissue, suspension cellcultures, immature inflorescence, shoot meristem, nodal explants, callustissue, hypocotyl tissue, cotyledons, roots, and leaves.

Any suitable plant culture medium can be used. Examples of suitablemedia would include but are not limited to MS-based media (Murashige andSkoog, Physiol. Plant, 15:473-497, 1962) or N6-based media (Chu et al.,Scientia Sinica 18:659, 1975) supplemented with additional plant growthregulators including but not limited to auxins, cytokinins, ABA, andgibberellins. Those of skill in the art are familiar with the variety oftissue culture media, which when supplemented appropriately, supportplant tissue growth and development and are suitable for planttransformation and regeneration. These tissue culture media can eitherbe purchased as a commercial preparation, or custom prepared andmodified. Those of skill in the art are aware that media and mediasupplements such as nutrients and growth regulators for use intransformation and regeneration and other culture conditions such aslight intensity during incubation, pH, and incubation temperatures thatcan be optimized for the particular variety of interest.

One of ordinary skill will appreciate that, after an expression cassetteis stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

Identification of Plants with an Altered Phenotype

An Arabidopsis activation tagging screen (ACTTAG) was used to identifythe association between 1) ACTTAG plant lines with an altered oil,protein and/or fiber content (see columns 4, 5 and 6 respectively, ofTable 1, below) and 2) the nucleic acid sequences identified in column 3of Tables 2 and 3, wherein each nucleic acid sequence is provided with agene alias or a HIO designation (HIO#; see column 1 in Tables 1, 2, and3). The HIO designation is arbitrary and does not necessarily relate toa plant having a high oil (HIO) phenotype.

Briefly, and as further described in the Examples, a large number ofArabidopsis plants were mutated with the pSKIO15 vector, which comprisesa T-DNA from the T1 plasmid of Agrobacterium tumifaciens, a viralenhancer element, and a selectable marker gene (Weigel et al., 2000,Plant Physiology, 122:1003-1013). When the T-DNA inserts into the genomeof transformed plants, the enhancer element can cause up-regulation ofgenes in the vicinity, generally within about nine kilobases (kb) of theenhancers. T1 plants were exposed to the selective agent in order tospecifically recover transformed plants that expressed the selectablemarker and therefore harbored T-DNA insertions. T1 plants were allowedto grow to maturity, self-fertilize and produce seed. T2 seed washarvested, labeled and stored. To amplify the seed stocks, abouteighteen T2 were sown in soil and, after germination, exposed to theselective agent to recover transformed T2 plants. T3 seed from theseplants was harvested and pooled. Oil, protein and fiber content of theseed were estimated using Near Infrared Spectroscopy (NIR) as describedin the Examples.

The association of a HIO nucleic acid sequence with an altered phenotypewas discovered by analysis of the genomic DNA sequence flanking theT-DNA insertion in the ACTTAG line identified in column 3 of Table 1. AnACTTAG line is a family of plants derived from a single plant that wastransformed with a T-DNA element containing four tandem copies of theCaMV 35S enhancers. Accordingly, the disclosed HIO nucleic acidsequences and/or polypeptides may be employed in the development oftransgenic plants having an altered, for example high oil, phenotype.HIO nucleic acid sequences may be used in the generation of transgenicplants, such as oilseed crops, that provide improved oil yield fromoilseed processing and result in an increase in the quantity of oilrecovered from seeds of the transgenic plant. HIO nucleic acid sequencesmay also be used in the generation of transgenic plants, such as feedgrain crops, that provide an altered phenotype resulting in increasedenergy for animal feeding, for example, seeds or seed meal with analtered protein and/or fiber content, resulting in an increase in AME.HIO nucleic acid sequences may further be used to increase the oilcontent of specialty oil crops, in order to augment yield and/orrecovery of desired unusual fatty acids. Specific non-limiting examplesof unusual fatty acids are ricinoleic acid, vernolic acid and the verylong chain polyunsaturated fatty acids docosahexaenoic acid (DHA) andeicosapentaenoic acid (EPA). Transgenic plants that have beengenetically modified to express HIO polypeptides can be used in theproduction of seeds, wherein the transgenic plants are grown, and oiland seed meal are obtained from plant parts (e.g. seed) using standardmethods.

HIO Nucleic Acids and Polypeptides

The HIO designation for each of the HIO nucleic acid sequencesdiscovered in the activation tagging screen described herein are listedin column 1 of Tables 1-3, below. The disclosed HIO polypeptides arelisted in column 4 of Tables 2 and 3, below. The HIO designation isarbitrary and does not necessarily relate to a plant having a high oil(HIO) phenotype. As used herein, the gene alias or HIO designationrefers to any polypeptide sequence (or the nucleic acid sequence thatencodes it) that when expressed in a plant causes an altered phenotypein any part of the plant, for example the seeds. In one embodiment, aHIO polypeptide refers to a full-length HIO protein, or a fragment,derivative (variant), or ortholog or paralog thereof that is“functionally active,” such that the protein fragment, derivative, orortholog or paralog exhibits one or more or the functional activitiesassociated with one or more of the disclosed full-length HIOpolypeptides, for example, the amino acid sequences provided in theGenBank entry referenced in column 4 of Table 2, and 3 which correspondto the amino acid sequences set forth as SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14,or an ortholog or paralog thereof. In one preferred embodiment, afunctionally active HIO polypeptide causes an altered phenotype in atransgenic plant. In another embodiment, a functionally active HIOpolypeptide causes an altered oil, protein, and/or fiber contentphenotype (for example, an altered seed meal content phenotype) whenmis-expressed in a plant. In other preferred embodiments, mis-expressionof the HIO polypeptide causes a high oil (such as, increased oil), highprotein (such as, increased total protein or digestible protein), and/orlow fiber (such as, decreased fiber) phenotype in a plant. In yet otherpreferred embodiments, mis-expression of the HIO polypeptide causesunchanged oil, high protein (such as, increased total protein ordigestible protein), and/or low fiber (such as, decreased fiber)phenotype in a plant. In another embodiment, mis-expression of the HIOpolypeptide causes an improved AME of meal. In yet another embodiment, afunctionally active HIO polypeptide can rescue defective (includingdeficient) endogenous HIO polypeptide activity when expressed in a plantor in plant cells; the rescuing polypeptide may be from the same or froma different species as the species with the defective polypeptideactivity. The disclosure also provides feed, meal, grain, food, or seedcomprising the HIO polypeptide, or a fragment, derivative (variant), orortholog or paralog thereof.

In another embodiment, a functionally active fragment of a full lengthHIO polypeptide (for example, a functionally active fragment of a nativepolypeptide having the amino acid sequence set forth as SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,or SEQ ID NO: 14, or a naturally occurring ortholog or paralog thereof)retains one or more of the biological properties associated with thefull-length HIO polypeptide, such as signaling activity, bindingactivity, catalytic activity, or cellular or extra-cellular localizingactivity. A HIO fragment preferably comprises a HIO domain, such as a C-or N-terminal or catalytic domain, among others, and preferablycomprises at least 10, preferably at least 20, more preferably at least25, and most preferably at least 50 contiguous amino acids of a HIOprotein. Functional domains of HIO genes are listed in column 6 of Table2 and can be identified using the PFAM program (Bateman A et al., 1999,Nucleic Acids Res. 27:260-262) or INTERPRO (Mulder et al., 2003, NucleicAcids Res. 31, 315-318) program. Functionally active variants offull-length HIO polypeptides, or fragments thereof, include polypeptideswith amino acid insertions, deletions, or substitutions that retain oneof more of the biological properties associated with the full-length HIOpolypeptide. In some cases, variants are generated that change thepost-translational processing of an HIO polypeptide. For instance,variants may have altered protein transport or protein localizationcharacteristics, or altered protein half-life, compared to the nativepolypeptide.

As used herein, the term “HIO nucleic acid” refers to any polynucleotidethat when expressed in a plant causes an altered phenotype in any partof the plant, for example the seeds. In one embodiment, a HIOpolynucleotide encompasses nucleic acids with the sequence provided inor complementary to the GenBank entry referenced in column 3 of Tables 2and 3, which correspond to nucleic acid sequences set forth as SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, or SEQ ID NO: 13, as well as functionally active fragments,derivatives, or orthologs or paralogs thereof. A HIO nucleic acid ofthis disclosure may be DNA, derived from genomic DNA or cDNA, or RNA.

In one embodiment, a functionally active HIO nucleic acid encodes or iscomplementary to a nucleic acid that encodes a functionally active HIOpolypeptide. A functionally active HIO nucleic acid also includesgenomic DNA that serves as a template for a primary RNA transcript(i.e., an mRNA precursor) that requires processing, such as splicing,before encoding the functionally active HIO polypeptide. A HIO nucleicacid can include other non-coding sequences, which may or may not betranscribed; such sequences include 5′ and 3′ UTRs, polyadenylationsignals and regulatory sequences that control gene expression, amongothers, as are known in the art. Some polypeptides require processingevents, such as proteolytic cleavage, covalent modification, etc., inorder to become fully active. Accordingly, functionally active nucleicacids may encode the mature or the pre-processed HIO polypeptide, or anintermediate form. A HIO polynucleotide can also include heterologouscoding sequences, for example, sequences that encode a marker includedto facilitate the purification of the fused polypeptide, or atransformation marker. In another embodiment, a functionally active HIOnucleic acid is capable of being used in the generation ofloss-of-function HIO phenotypes, for instance, via antisensesuppression, co-suppression, etc. The disclosure also provides feed,meal, grain, food, or seed comprising a nucleic acid sequence thatencodes an HIO polypeptide.

In one preferred embodiment, a HIO nucleic acid used in the disclosedmethods comprises a nucleic acid sequence that encodes, or iscomplementary to a sequence that encodes, a HIO polypeptide having atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity to a disclosed HIO polypeptide sequence, for example the aminoacid sequence set forth as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.

In another embodiment, a HIO polypeptide comprises a polypeptidesequence with at least 50% or 60% identity to a disclosed HIOpolypeptide sequence (for example, the amino acid sequence set forth asSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, or SEQ ID NO: 14) and may have at least 70%, 80%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity to a disclosed HIOpolypeptide sequence. In a further embodiment, a HIO polypeptidecomprises 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity to a disclosed HIO polypeptide sequence, and may include aconserved protein domain of the HIO polypeptide (such as the proteindomain(s) listed in column 6 of Table 2). In another embodiment, a HIOpolypeptide comprises a polypeptide sequence with at least 50%, 60%,70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to afunctionally active fragment of the polypeptide referenced in column 4of Table 2. In yet another embodiment, a HIO polypeptide comprises apolypeptide sequence with at least 50%, 60%, 70%, 80%, 90%, 95%, 97%,98%, or 99% identity to the polypeptide sequence of the GenBank entryreferenced in column 4 of Table 2 over its entire length and comprises aconserved protein domain(s) listed in column 6 of Table 2.

In another aspect, a HIO polynucleotide sequence is at least 50% to 60%identical over its entire length to a disclosed HIO nucleic acidsequence, such as the nucleic acid sequence set forth as SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1, orSEQ ID NO: 13, or nucleic acid sequences that are complementary to sucha HIO sequence, and may comprise at least 70%, 80%, 85%, 90%, 95%, 97%,98%, or 99% sequence identity to the disclosed HIO sequence, or afunctionally active fragment thereof, or complementary sequences. Inanother embodiment, a disclosed HIO nucleic acid comprises a nucleicacid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1, or SEQ ID NO: 13, or nucleic acidsequences that are complementary to such a HIO sequence, and nucleicacid sequences that have substantial sequence homology to a such HIOsequences. As used herein, the phrase “substantial sequence homology”refers to those nucleic acid sequences that have slight orinconsequential sequence variations from such HIO sequences, i.e., thesequences function in substantially the same manner and encode an HIOpolypeptide.

As used herein, “percent (%) sequence identity” with respect to aspecified subject sequence, or a specified portion thereof, is definedas the percentage of nucleotides or amino acids in an identifiedsequence identical with the nucleotides or amino acids in the subjectsequence (or specified portion thereof), after aligning the sequencesand introducing gaps, if necessary to achieve the maximum percentsequence identity, as generated by the program WU-BLAST-2.0a19 (Altschulet al., J. Mol. Biol., 1990, 215:403-410) with search parameters set todefault values. The HSP S and HSP S2 parameters are dynamic values andare established by the program itself depending upon the composition ofthe particular sequence and composition of the particular databaseagainst which the sequence of interest is being searched. A “percent (%)identity value” is determined by the number of matching identicalnucleotides or amino acids divided by the sequence length for which thepercent identity is being reported. “Percent (%) amino acid sequencesimilarity” is determined by performing the same calculation as fordetermining % amino acid sequence identity, but including conservativeamino acid substitutions in addition to identical amino acids in thecomputation. A conservative amino acid substitution is one in which anamino acid is substituted for another amino acid having similarproperties such that the folding or activity of the protein is notsignificantly affected. Aromatic amino acids that can be substituted foreach other are phenylalanine, tryptophan, and tyrosine; interchangeablehydrophobic amino acids are leucine, isoleucine, methionine, and valine;interchangeable polar amino acids are glutamine and asparagine;interchangeable basic amino acids are arginine, lysine and histidine;interchangeable acidic amino acids are aspartic acid and glutamic acid;and interchangeable small amino acids are alanine, serine, threonine,cysteine and glycine.

Derivative nucleic acid molecules of the subject nucleic acid moleculesinclude sequences that selectively hybridize to the disclosed HIOnucleic acid sequences (for example, the nucleic acid sequence set forthas SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, or SEQ ID NO: 13). The stringency of hybridization can becontrolled by temperature, ionic strength, pH, and the presence ofdenaturing agents such as formamide during hybridization and washing.Conditions routinely used are well known (see, e.g., Current Protocol inMolecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers(1994); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual(Second Edition), Cold Spring Harbor Press, Plainview, N.Y.,).

In some embodiments, a nucleic acid molecule of the disclosure iscapable of hybridizing to a nucleic acid molecule containing thedisclosed nucleotide sequence under stringent hybridization conditionsthat are: prehybridization of filters containing nucleic acid for 8hours to overnight at 65° C. in a solution comprising 6× single strengthcitrate (SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0),5×Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/md herringsperm DNA; hybridization for 18-20 hours at 65° C. in a solutioncontaining 6×SSC, 1×Denhardt's solution, 100 μg/ml yeast tRNA and 0.05%sodium pyrophosphate; and washing of filters at 65° C. for 1 h in asolution containing 0.1×SSC and 0.1% SDS (sodium dodecyl sulfate). Inother embodiments, moderately stringent hybridization conditions areused that are: pretreatment of filters containing nucleic acid for 6 hat 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl(pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/mldenatured salmon sperm DNA; hybridization for 18-20 h at 40° C. in asolution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mMEDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at55° C. in a solution containing 2×SSC and 0.1% SDS. Alternatively, lowstringency conditions can be used that comprise: incubation for 8 hoursto overnight at 37° C. in a solution comprising 20% formamide, 5×SSC, 50mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured sheared salmon sperm DNA; hybridizationin the same buffer for 18 to 20 hours; and washing of filters in 1×SSCat about 37° C. for 1 hour.

As a result of the degeneracy of the genetic code, a number ofpolynucleotide sequences encoding a HIO polypeptide can be produced. Forexample, codons may be selected to increase the rate at which expressionof the polypeptide occurs in a particular host species, in accordancewith the optimum codon usage dictated by the particular host organism(see, e.g., Nakamura et al., 1999, Nucleic Acids Res. 27:292). Suchsequence variants may be used in the methods disclosed herein.

The disclosed methods may use orthologs (and/or paralogs) of a disclosedArabidopsis HIO nucleic acid sequence. Representative putative orthologs(and/or paralogs) of each of the disclosed Arabidopsis HIO genes areidentified in column 5 of Table 3, below. Methods of identifying theorthologs in other plant species are known in the art. In general,orthologs in different species retain the same function, due to presenceof one or more protein motifs and/or 3-dimensional structures. Inevolution, when a gene duplication event follows speciation, a singlegene in one species, such as Arabidopsis, may correspond to multiplegenes (paralogs) in another. When sequence data is available for aparticular plant species, orthologs are generally identified by sequencehomology analysis, such as BLAST analysis, usually using protein baitsequences. Sequences are assigned as a potential ortholog if the besthit sequence from the forward BLAST result retrieves the original querysequence in the reverse BLAST (Huynen M A and Bork P, 1998, Proc. Natl.Acad. Sci., 95:5849-5856; Huynen M A et al., 2000, Genome Research,10:1204-1210).

Programs for multiple sequence alignment, such as CLUSTAL (Thompson J Det al., 1994, Nucleic Acids Res. 22:4673-4680) may be used to highlightconserved regions and/or residues of homologous (orthologous and/orparalogous) proteins and to generate phylogenetic trees. In aphylogenetic tree representing multiple homologous sequences fromdiverse species (e.g., retrieved through BLAST analysis), orthologoussequences from two species generally appear closest on the tree withrespect to all other sequences from these two species. Structuralthreading or other analysis of protein folding (e.g., using software byProCeryon, Biosciences, Salzburg, Austria) may also identify potentialorthologs. Nucleic acid hybridization methods may also be used to findorthologous genes and are preferred when sequence data are notavailable. Degenerate PCR and screening of cDNA or genomic DNA librariesare common methods for finding related gene sequences and are well knownin the art (see, e.g., Sambrook, 1989, Molecular Cloning: A LaboratoryManual (Second Edition), Cold Spring Harbor Press, Plainview, N.Y.;Dieffenbach and Dveksler, 1995, PCR Primer: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY). For instance, methods forgenerating a cDNA library from the plant species of interest and probingthe library with partially homologous gene probes are described inSambrook et al. A highly conserved portion of the Arabidopsis HIO codingsequence may be used as a probe. HIO ortholog nucleic acids mayhybridize to the nucleic acid of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 underhigh, moderate, or low stringency conditions. After amplification orisolation of a segment of a putative ortholog, that segment may becloned and sequenced by standard techniques and utilized as a probe toisolate a complete cDNA or genomic DNA clone.

Alternatively, it is possible to initiate an EST project to generate adatabase of sequence information for the plant species of interest. Inanother approach, antibodies that specifically bind known HIOpolypeptides are used for ortholog (and/or paralog) isolation (see,e.g., Harlow and Lane, 1988, 1999, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, New York). Western blot analysis candetermine that a HIO ortholog (i.e., a protein orthologous to adisclosed HIO polypeptide) is present in a crude extract of a particularplant species. When reactivity is observed, the sequence encoding thecandidate ortholog may be isolated by screening expression librariesrepresenting the particular plant species. Expression libraries can beconstructed in a variety of commercially available vectors, includinglambda gt11, as described in Sambrook, et al., 1989. Once the candidateortholog(s) are identified by any of these means, candidate orthologoussequence are used as bait (the “query”) for the reverse BLAST againstsequences from Arabidopsis or other species in which HIO nucleic acidand/or polypeptide sequences have been identified.

HIO nucleic acids and polypeptides may be obtained using any availablemethod. For instance, techniques for isolating cDNA or genomic DNAsequences of interest by screening DNA libraries or by using polymerasechain reaction (PCR), as previously described, are well known in theart. Alternatively, nucleic acid sequence may be synthesized. Any knownmethod, such as site directed mutagenesis (Kunkel T A et al., 1991,Methods Enzymol. 204:125-39), may be used to introduce desired changesinto a cloned nucleic acid.

In general, the methods disclosed herein involve incorporating thedesired form of the HIO nucleic acid into a plant expression vector fortransformation of plant cells, and the HIO polypeptide is expressed inthe host plant. Transformed plants and plant cells expressing an HIOpolypeptide express an altered phenotype and, in one specific,non-limiting example, may have high (increased) oil, high (increased)protein, and/or low (decreased) fiber content.

An “isolated” HIO nucleic acid molecule is other than in the form orsetting in which it is found in nature, and is identified and separatedfrom least one contaminant nucleic acid molecule with which it isordinarily associated in the natural source of the HIO nucleic acid.However, an isolated HIO nucleic acid molecule includes HIO nucleic acidmolecules contained in cells that ordinarily express the HIO polypeptidewhere, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

Generation of Genetically Modified Plants with an Altered Phenotype

The disclosed HIO nucleic acids and polypeptides may be used in thegeneration of transgenic plants having a modified or altered phenotype,for example an altered oil, protein, and/or fiber content phenotype. Asused herein, an “altered oil content (phenotype)” may refer to alteredoil content in any part of the plant. In a preferred embodiment, alteredexpression of the HIO gene in a plant is used to generate plants with ahigh oil content (phenotype). As used herein, an “altered total proteincontent (phenotype)” or an “altered digestible protein content(phenotype)” may refer to altered protein (total or digestible) contentin any part of the plant. In a preferred embodiment, altered expressionof the HIO gene in a plant is used to generate plants with a high (orincreased) total or digestible protein content (phenotype). As usedherein, an “altered fiber content (phenotype)” may refer to alteredfiber content in any part of the plant. In a preferred embodiment,altered expression of the HIO gene in a plant is used to generate plantswith a low (or decreased) fiber content (phenotype). The altered oil,protein and/or fiber content is often observed in seeds. Examples of atransgenic plant include plants comprising a plant transformation vectorwith a nucleotide sequence that encodes or is complementary to asequence that encodes an HIO polypeptide having the amino acid sequenceas set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, or an ortholog orparalog thereof.

Transgenic plants, such as corn, soybean and canola containing thedisclosed nucleic acid sequences, can be used in the production ofvegetable oil and meal. Vegetable oil is used in a variety of foodproducts, while meal from seed is used as an animal feed. Afterharvesting seed from transgenic plants, the seed is cleaned to removeplant stalks and other material and then flaked in roller mills to breakthe hulls. The crushed seed is heated to 75-100° C. to denaturehydrolytic enzymes, lyse the unbroken oil containing cells, and allowsmall oil droplets to coalesce. Most of the oil is then removed (and canbe recovered) by pressing the seed material in a screw press. Theremaining oil is removed from the presscake by extraction with andorganic solvents, such as hexane. The solvent is removed from the mealby heating it to approximately 100° C. After drying, the meal is thengranulated to a consistent form. The meal, containing the protein,digestible carbohydrate, and fiber of the seed, may be mixed with othermaterials prior to being used as an animal feed.

The methods described herein for generating transgenic plants aregenerally applicable to all plants. Although activation tagging and geneidentification is carried out in Arabidopsis, the HIO nucleic acidsequence (or an ortholog, paralog, variant or fragment thereof) may beexpressed in any type of plant. In a preferred embodiment, oil-producingplants produce and store triacylglycerol in specific organs, primarilyin seeds. Such species include soybean (Glycine max), rapeseed andcanola (including Brassica napus, B. campestris), sunflower (Helianthusannus), cotton (Gossypium hirsutum), corn (Zea mays), cocoa (Theobromacacao), safflower (Carthamus tinctorius), oil palm (Elaeis guineensis),coconut palm (Cocos nucifera), flax (Linum usitatissimum), castor(Ricinus communis), and peanut (Arachis hypogaea), as well as wheat,rice and oat. Fruit- and vegetable-bearing plants, grain-producingplants, nut-producing plants, rapid cycling Brassica species, alfalfa(Medicago sativa), tobacco (Nicotiana), turfgrass (Poaceae family),other forage crops, and wild species may also be a source of uniquefatty acids. In other embodiments, any plant expressing the HIO nucleicacid sequence can also express increased protein and/or decreased fibercontent in a specific plant part or organ, such as in seeds.

The skilled artisan will recognize that a wide variety of transformationtechniques exist in the art, and new techniques are continually becomingavailable. Any technique that is suitable for the target host plant canbe employed within the scope of the present invention. For example, theconstructs can be introduced in a variety of forms including, but notlimited to, as a strand of DNA, in a plasmid, or in an artificialchromosome. The introduction of the constructs into the target plantcells can be accomplished by a variety of techniques, including, but notlimited to, Agrobacterium-mediated transformation, electroporation,microinjection, microprojectile bombardment, calcium-phosphate-DNAco-precipitation, or liposome-mediated transformation of a heterologousnucleic acid. The transformation of the plant is preferably permanent,i.e. by integration of the introduced expression constructs into thehost plant genome, so that the introduced constructs are passed ontosuccessive plant generations. Depending upon the intended use, aheterologous nucleic acid construct comprising an HIO polynucleotide mayencode the entire protein or a biologically active portion thereof.

In one embodiment, binary Ti-based vector systems may be used totransfer polynucleotides. Standard Agrobacterium binary vectors areknown to those of skill in the art, and many are commercially available(e.g., pBI121 Clontech Laboratories, Palo Alto, Calif.). A construct orvector may include a plant promoter to express the nucleic acid moleculeof choice. In a preferred embodiment, the promoter is a plant promoter.

The optimal procedure for transformation of plants with Agrobacteriumvectors will vary with the type of plant being transformed. Exemplarymethods for Agrobacterium-mediated transformation include transformationof explants of hypocotyl, shoot tip, stem or leaf tissue, derived fromsterile seedlings and/or plantlets. Such transformed plants may bereproduced sexually, or by cell or tissue culture. Agrobacteriumtransformation has been previously described for a large number ofdifferent types of plants and methods for such transformation may befound in the scientific literature. Of particular relevance are methodsto transform commercially important crops, such as plants of theBrassica species, including canola and rapeseed, (De Block et al., 1989,Plant Physiol., 91:694-701), maize (Ishida et al., 1996 NatureBiotechnol. 14:745-750, Zhang et al., 2002 Plant Cell Rep. 21:263-270)sunflower (Everett et al., 1987, Bio/Technology, 5:1201), soybean(Christou et al., 1989, Proc. Natl. Acad. Sci. USA, 86:7500-7504; Klineet al., 1987, Nature, 327:70), wheat, rice and oat.

Expression (including transcription and translation) of a HIO nucleicacid sequence may be regulated with respect to the level of expression,the tissue type(s) where expression takes place and/or developmentalstage of expression. A number of heterologous regulatory sequences(e.g., promoters and enhancers) are available for controlling theexpression of a HIO nucleic acid. These include constitutive, inducibleand regulatable promoters, as well as promoters and enhancers thatcontrol expression in a tissue- or temporal-specific manner. Exemplaryconstitutive promoters include the raspberry E4 promoter (U.S. Pat. Nos.5,783,393 and 5,783,394), the nopaline synthase (NOS) promoter (Ebert etal., Proc. Natl. Acad. Sci. (U.S.A.) 84:5745-5749, 1987), the octopinesynthase (OCS) promoter (which is carried on tumor-inducing plasmids ofAgrobacterium tumefaciens), the caulimovirus promoters such as thecauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Mol.Biol. 9:315-324, 1987) and the CaMV 35S promoter (Odell et al., Nature313:810-812, 1985 and Jones J D et al, 1992, Transgenic Res.,1:285-297), the figwort mosaic virus 35S-promoter (U.S. Pat. No.5,378,619), the light-inducible promoter from the small subunit ofribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), the Adh promoter(Walker et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:6624-6628, 1987), thesucrose synthase promoter (Yang et al., Proc. Natl. Acad. Sci. (U.S.A.)87:4144-4148, 1990), the R gene complex promoter (Chandler et al., ThePlant Cell 1:1175-1183, 1989), the chlorophyll a/b binding protein genepromoter, the CsVMV promoter (Verdaguer B et al., 1998, Plant Mol.Biol., 37:1055-1067), and the melon actin promoter (published PCTapplication WO0056863). Exemplary tissue-specific promoters include thetomato E4 and E8 promoters (U.S. Pat. No. 5,859,330) and the tomato 2AIIgene promoter (Van Haaren M J J et al., 1993, Plant Mol. Bio.,21:625-640).

In one preferred embodiment, expression of the HIO nucleic acid sequenceis under control of regulatory sequences from genes whose expression isassociated with early seed and/or embryo development. Indeed, in apreferred embodiment, the promoter used is a seed-enhanced promoter.Examples of such promoters include the 5′ regulatory regions from suchgenes as napin (Kridl et al., Seed Sci. Res. 1:209:219, 1991), globulin(Belanger and Kriz, Genet., 129: 863-872, 1991, GenBank Accession No.L22295), gamma zein Z 27 (Lopes et al., Mol Gen Genet., 247:603-613,1995), L3 oleosin promoter (U.S. Pat. No. 6,433,252), phaseolin (Bustoset al., Plant Cell, 1(9):839-853, 1989), arcelin5 (U.S. Application No.2003/0046727), a soybean 7S promoter, a 7Sα promoter (U.S. ApplicationNo. 2003/0093828), the soybean 7Sα′ beta conglycinin promoter, a 7Sα′promoter (Beachy et al., EMBO J., 4:3047, 1985; Schuler et al., NucleicAcid Res., 10(24):8225-8244, 1982), soybean trypsin inhibitor (Riggs etal., Plant Cell 1(6):609-621, 1989), ACP (Baerson et al., Plant Mol.Biol., 22 (2):255-267, 1993), stearoyl-ACP desaturase (Slocombe et al.,Plant Physiol. 104(4):167-176, 1994), soybean a′ subunit ofβ-conglycinin (Chen et al., Proc. Natl. Acad. Sci. 83:8560-8564, 1986),Viciafaba USP (P-Vf.Usp, SEQ ID NO: 1, 2, and 3 in (U.S. Application No.2003/229918) and Zea mays L3 oleosin promoter (Hong et al., Plant Mol.Biol., 34(3):549-555, 1997). Also included are the zeins, which are agroup of storage proteins found in corn endosperm. Genomic clones forzein genes have been isolated (Pedersen et al., Cell, 29:1015-1026,1982; and Russell et al., Transgenic Res. 6(2):157-168) and thepromoters from these clones, including the 15 kD, 16 kD, 19 kD, 22 kD,27 kD and genes, could also be used. Other promoters known to function,for example, in corn include the promoters for the following genes:waxy, Brittle, Shrunken 2, Branching enzymes I and II, starch synthases,debranching enzymes, oleosins, glutelins and sucrose synthases. Legumegenes whose promoters are associated with early seed and embryodevelopment include V. faba leguin (Baumlein et al., 1991, Mol. Gen.Genet. 225:121-8; Baumlein et al., 1992, Plant J. 2:233-9), V. faba usp(Fiedler et al., 1993, Plant Mol. Biol. 22:669-79), pea convicilin (Bownet al., 1988, Biochem. J. 251:717-26), pea/ectin (dePater et al., 1993,Plant Cell 5:877-86), P. vulgaris beta phaseolin (Bustos et al., 1991,EMBO J. 10: 1469-79), P. vulgaris DLEC2 and PHS [beta] (Bobb et al.,1997, Nucleic Acids Res. 25:641-7), and soybean beta-Conglycinin, 7Sstorage protein (Chamberland et al., 1992, Plant Mol. Biol. 19:937-49).

Cereal genes whose promoters are associated with early seed and embryodevelopment include rice glutelin (“GluA-3,” Yoshihara and Takaiwa,1996, Plant Cell Physiol. 37:107-11; “GluB-1,” Takaiwa et al., 1996,Plant Mol. Biol. 30:1207-21; Washida et al., 1999, Plant Mol. Biol.40:1-12; “Gt3,” Leisy et al., 1990, Plant Mol. Biol. 14:41-50), riceprolamin (Zhou & Fan, 1993, Transgenic Res. 2:141-6), wheat prolamin(Hammond-Kosack et al., 1993, EMBO J. 12:545-54), maize zein (Z4, Matzkeet al., 1990, Plant Mol. Biol. 14:323-32), and barley B-hordeins(Entwistle et al., 1991, Plant Mol. Biol. 17:1217-31).

Other genes whose promoters are associated with early seed and embryodevelopment include oil palm GLO7A (7S globulin, Morcillo et al., 2001,Physiol. Plant 112:233-243), Brassica napus napin, 2S storage protein,and napA gene (Josefsson et al., 1987, J. Biol. Chem. 262:12196-201;Stalberg et al., 1993, Plant Mol. Biol. 1993 23:671-83; Ellerstrom etal., 1996, Plant Mol. Biol. 32:1019-27), Brassica napus oleosin (Keddieet al., 1994, Plant Mol. Biol. 24:327-40), Arabidopsis oleosin (Plant etal., 1994, Plant Mol. Biol. 25:193-205), Arabidopsis FAE1 (Rossak etal., 2001, Plant Mol. Biol. 46:717-25), Canavalia gladiata conA(Yamamoto et al., 1995, Plant Mol. Biol. 27:729-41), and Catharanthusrose us strictosidine synthase (Str, Ouwerkerk and Memelink, 1999, Mol.Gen. Genet. 261:635-43). In another preferred embodiment, regulatorysequences from genes expressed during oil biosynthesis are used (see,e.g., U.S. Pat. No. 5,952,544). Alternative promoters are from plantstorage protein genes (Bevan et al., 1993, Philos. Trans. R. Soc. Lond.B. Biol. Sci. 342:209-15). Additional promoters that may be utilized aredescribed, for example, in U.S. Pat. Nos. 5,378,619; 5,391,725;5,428,147; 5,447,858; 5,608,144; 5,608,144; 5,614,399; 5,633,441;5,633,435; and 4,633,436.

In another embodiment, the endogenous HIO gene may be placed under thecontrol of a transgenic transcription factor or used to design bindingsites that modulates its expression. One such class of transcriptionfactors are the Cys₂-His₂-zinc finger proteins (ZFPs). ZFPs are commonDNA binding proteins and can be designed to specifically bind tospecific DNA sequences (Beerli & Barbas, Nat. Biotechnol., 2002,20:135-141.; Gommans et al., J Mol. Biol., 2005, 354:507-519).Individual zinc-finger domains are composed of approximately 30 aminoacids, are structurally conserved and can interact with 3-4 bp of DNA. Apolypeptide containing multiple zinc-fingers designed to bind to aspecific DNA sequence in the promoter of a HIO gene can be synthesized.The principles for designing the zinc finger domains to interact withspecific DNA sequences have been described in Segal et al., (Segal etal., Proc Natl Acad Sci USA., 1999, 96:2758-2763), Dreier et al. (Dreieret al., J Mol Biol., 2000, 303:489-502), and Beerli and Barbas (Beerli &Barbas, Nat. Biotechnol., 2002, 20:135-141). These DNA binding domainsmay be fused to effector domains to form a synthetic ZFP that mayregulate transcription of genes to which they bind. Effector domainsthat can activate transcription include but are not limited to theacidic portion of the herpes simplex virus protein VP16 (Sadowski etal., Nature., 1988, 335:563-564) and VP64 (Beerli et al., Proc Natl AcadSci USA., 1998, 95:14628-14633), and the NF-κB transcription factor p65domain (Bae et al., Nat. Biotechnol., 2003, 21:275-280., Liu et al., JBiol Chem., 2001, 276:11323-11334). Effector domains that can represstranscription include but are not limited to mSIN3 and KRAB (Ayer etal., Mol Cell Biol., 1996, 16:5772-5781, Beerli & Barbas, Nat.Biotechnol., 2002, 20:135-141, Beerli et al., Proc Natl Acad Sci USA,1998, 95:14628-14633, Margolin et al., Proc Natl Acad Sci USA., 1994,91:4509-4513). These approaches have been shown to work in plants (Guanet al., Proc Natl Acad Sci USA., 2002, 99:13296-13301, Stege et al.,Plant J., 2002, 32:1077-1086, Van Eenennaam et al., Metab Eng., 2004,6:101-108).

In yet another aspect, in some cases it may be desirable to inhibit theexpression of the endogenous HIO nucleic acid sequence in a host cell.Exemplary methods for practicing this aspect of the invention include,but are not limited to antisense suppression (Smith, et al., 1988,Nature, 334:724-726; van der Krol et al., 1988, BioTechniques,6:958-976); co-suppression (Napoli, et al., 1990, Plant Cell,2:279-289); ribozymes (PCT Publication WO 97/10328); and combinations ofsense and antisense (Waterhouse, et al., 1998, Proc. Natl. Acad. Sci.USA, 95:13959-13964). Methods for the suppression of endogenoussequences in a host cell typically employ the transcription ortranscription and translation of at least a portion of the sequence tobe suppressed. Such sequences may be homologous to coding as well asnon-coding regions of the endogenous sequence. Antisense inhibition mayuse the entire cDNA sequence (Sheehy et al., 1988, Proc. Natl. Acad.Sci. USA, 85:8805-8809), a partial cDNA sequence including fragments of5′ coding sequence, (Cannon et al., 1990, Plant Mol. Biol., 15:39-47),or 3′ non-coding sequences (Ch'ng et al., 1989, Proc. Natl. Acad. Sci.USA, 86:10006-10010). Cosuppression techniques may use the entire cDNAsequence (Napoli et al., 1990, Plant Cell, 2:279-289; van der Krol etal., 1990, Plant Cell, 2:291-299), or a partial cDNA sequence (Smith etal., 1990, Mol. Gen. Genetics, 224:477-481).

Standard molecular and genetic tests may be performed to further analyzethe association between a nucleic acid sequence and an observedphenotype. Exemplary techniques are described below.

1. DNA/RNA Analysis

The stage- and tissue-specific gene expression patterns in mutant versuswild-type lines may be determined, for instance, by in situhybridization. Analysis of the methylation status of the gene,especially flanking regulatory regions, may be performed. Other suitabletechniques include over-expression, ectopic expression, expression inother plant species and gene knock-out (reverse genetics, targetedknock-out, viral induced gene silencing (VIGS; see, Baulcombe D, 1999,Arch. Virol. Suppl. 15:189-201).

In a preferred application expression profiling, generally by microarrayanalysis, is used to simultaneously measure differences or inducedchanges in the expression of many different genes. Techniques formicroarray analysis are well known in the art (Schena M et al., Science1995 270:467-470; Baldwin D et al., 1999, Cur. Opin. Plant Biol.2(2):96-103; Dangond F, Physiol Genomics (2000) 2:53-58; van Hal N L etal., J Biotechnol. (2000) 78:271-280; Richmond T and Somerville S, Curr.Opin. Plant Biol. 2000 3:108-116). Expression profiling of individualtagged lines may be performed. Such analysis can identify other genesthat are coordinately regulated as a consequence of the over-expressionof the gene of interest, which may help to place an unknown gene in aparticular pathway.

2. Gene Product Analysis

Analysis of gene products may include recombinant protein expression,antisera production, immunolocalization, biochemical assays forcatalytic or other activity, analysis of phosphorylation status, andanalysis of interaction with other proteins via yeast two-hybrid assays.

3. Pathway Analysis

Pathway analysis may include placing a gene or gene product within aparticular biochemical, metabolic or signaling pathway based on itsmis-expression phenotype or by sequence homology with related genes.Alternatively, analysis may comprise genetic crosses with wild-typelines and other mutant lines (creating double mutants) to order the genein a pathway, or determining the effect of a mutation on expression ofdownstream “reporter” genes in a pathway.

Generation of Mutated Plants with an Altered Phenotype

Additional methods are disclosed herein of generating a plant having analtered phenotype, wherein a plant is identified that has a mutation oran allele in its HIO nucleic acid sequence that results in an alteredphenotype, compared to plants lacking the mutation or allele. Themutated plant can be generated using one or more mutagens, for example achemical mutagen (such as ethylmethane sulfonate, methyl methanesulfonate, diethylsulfate, and nitrosoguanidine, or5-bromo-deoxyuridine) radiation, or ultraviolet light. In someembodiments of the method, the mutated plant can be bred to generateprogeny, which inherit the mutation or allele and have an alteredphenotype. For example, provided herein is a method of identifyingplants that have one or more mutations in the endogenous HIO nucleicacid sequence that confer an altered phenotype and generating progeny ofthese mutated plants having such a phenotype that are not transgenic.The mutated plants with an altered phenotype can have an altered oil,protein, and/or fiber content, or an altered seed meal content.

In one specific embodiment of the method, called “TILLING” (fortargeting induced local lesions in genomes), mutations are induced inthe seed of a plant of interest, for example, using EMS (ethylmethanesulfonate) treatment. The resulting plants are grown andself-fertilized, and the progeny are used to prepare DNA samples. PCRamplification and sequencing of the HIO nucleic acid sequence is used toidentify whether a mutated plant has a mutation in the HIO nucleic acidsequence. Plants having HIO mutations may then be tested for alteredoil, protein, and/or fiber content. To confirm that the HIO mutationcauses the modified phenotype, experiments correlating the presence ofthe modified gene and the modified phenotype through genetic crosses canbe performed. TILLING can identify mutations that alter the expressionof specific genes or the activity of proteins encoded by these genes(see Colbert et al., 2001, Plant Physiol. 126:480-484; McCallum et al.,2000, Nature Biotechnology 18:455-457).

In another specific embodiment of the method, a candidategene/Quantitative Trait Locus (QTLs) approach can be used in amarker-assisted breeding program to identify alleles of or mutations inthe HIO nucleic acid sequence or orthologs (and/or paralogs) of the HIOnucleic acid sequence that may confer altered oil, protein, and/or fibercontent (see Bert et al., Theor Appl Genet., 2003 June; 107(1):181-9;and Lionneton et al., Genome, 2002 December; 45(6):1203-15). Thus, in afurther aspect of the disclosure, a HIO nucleic acid is used to identifywhether a plant having altered oil, protein, and/or fiber content has amutation in an endogenous HIO nucleic acid sequence or has a particularallele that causes altered oil, protein, and/or fiber content in theplant.

While the disclosure has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from thedisclosure. All publications cited herein are expressly incorporatedherein by reference for the purpose of describing and disclosingcompositions and methodologies that might be used in connection with thedisclosure. All cited patents, patent applications, and sequenceinformation in referenced public databases are also incorporated byreference.

EXAMPLES Example 1 Generation of Plants with a HIO Phenotype byTransformation with an Activation Tagging Construct

This Example describes the generation of transgenic plants with alteredoil, protein, and/or fiber content.

Mutants were generated using the activation tagging “ACTTAG” vector,pSKI015 (GI#6537289; Weigel D et al., 2000, Plant Physiology,122:1003-1013). Standard methods were used for the generation ofArabidopsis transgenic plants, and were essentially as described inpublished application PCT WO0183697. Briefly, T0 Arabidopsis (Col-0)plants were transformed with Agrobacterium carrying the pSKI015 vector,which comprises T-DNA derived from the Agrobacterium T1 plasmid, anherbicide resistance selectable marker gene, and the 4×CaMV 35S enhancerelement. Transgenic plants were selected at the T1 generation based onherbicide resistance. T2 seed (from T1 plants) was harvested and sown insoil. T2 plants were exposed to the herbicide to kill plants lacking theACTTAG vector. T2 plants were grown to maturity, allowed toself-fertilize and set seed. T3 seed (from the T2 plants) was harvestedin bulk for each line.

T3 seed was analyzed by Near Infrared Spectroscopy (NIR) at the time ofharvest. NIR spectra were captured using a Bruker 22 near infraredspectrometer. Bruker Software was used to estimate total seed oil, totalseed protein and total seed fiber content using data from NIR analysisand reference methods according to the manufacturer's instructions. Oilcontent predicting calibrations were developed following the generalmethod of AOCS Procedure Aml-92, Official Methods and RecommendedPractices of the American Oil Chemists Society, 5th Ed., AOCS,Champaign, Ill. A NIR total protein content predicting calibration wasdeveloped using total nitrogen content data of seed samples followingthe general method of Dumas Procedure AOAC 968.06 (Official Methods ofAnalysis of AOAC International 17^(th) Edition AOAC, Gaithersburg, Md.).A NIR fiber content predicting calibration was developed using crudefiber content data of seed samples following the general method of AOACOfficial Method 962.09 (Official Methods of Analysis of AOACInternational 17^(th) Edition AOAC, Gaithersburg, Md.). A NIR oleic acidcontent predicting calibration was developed using oleic acid contentdata of seed samples determined by following the method of Browse et al.(1986 Anal. Biochem. 152:141-145). A NIR calibration curve forpredicting digestible protein content was developed by measuringdigestible protein content in a set of seed samples. Total proteincontent of in a known mass of seed was determined by measuring the totalnitrogen content of the seed using the Dumas method (AOAC OfficialMethod 968.06). The seed fiber is extracted from a separate seed sampleusing the method of Honig and Rackis, (1979, J. Agri. Food Chem., 27:1262-1266). The undigested protein remaining associated with the fiberis measured by the Dumas method (AOAC Official Method 968.06).Digestible protein content is determined by subtracting the amount ofundigested protein associated with the fiber from the total amount ofprotein in the seed.

Oil, protein and fiber predictions from NIR spectra were compared for82,274 individual ACTTAG lines. Subsequent to seed compositionalanalysis, the position of the ACTTAG element in the genome in each linewas determined by inverse PCR and sequencing. 37,995 lines withrecovered flanking sequences were considered in this analysis.

Seed oil, and protein values in 82,274 lines were determined by NIRspectroscopy and normalized to allow comparison of seed component valuesin plants grown at different times. Oil, protein and fiber values werenormalized by calculating the average oil, protein and fiber values inseed from all plants planted on the same day (including a large numberof other ACTTAG plants, including control, wild-type, or non-transgenicplants). The seed components for each line was expressed as a “percentrelative value” which was calculated by dividing the component value foreach line with the average component value for all lines planted on thesame day (which should approximate the value in control, wild-type, ornon-transgenic plants). The “percent relative protein” and “percentrelative fiber” were calculated similarly.

Inverse PCR was used to recover genomic DNA flanking the T-DNAinsertion. The PCR product was subjected to sequence analysis and placedon the genome using a basic BLASTN search and/or a search of theArabidopsis Information Resource (TAIR) database (available at thepublicly available website). Generally, promoters within 9 kb of theenhancers in the ACTTAG element are considered to be within “activationspace.” Genes with T-DNA inserts within coding sequences were notconsidered to be within “activation space.” The ACTTAG lines identifiedare listed in column 3 of Table 1. In some cases more than one ACTTAGline is associated with a gene. The relative oil, protein, fiber andoleic acid values in columns 4, 5, 6 and 7, respectively, are determinedby comparing the seed component in the plant identified in column 3relative to other plants grown at the same time and not displaying thetrait.

TABLE 1 4. Relative 5. Relative 6. Relative 7. Relative 1. Alias 2. TAIRID 3. Plant ID Oil (%) Protein (%) Fiber (%) Oleic Acid HIO2019 AAt1g17920 IN081493 118.99 95.25 100.45 102.12 HIO2019 A At1g17920IN006312 109.05 95.89 108.83 109.05 HIO2046 A At5g59010 IN089660 128.91142.4 46.44 HIO2046 A At5g59010 IN047347 102.37 104.89 96.13 106.83HIO2079 A At1g71410 IN083604 131.01 83.85 99.94 139.75 HIO2079 AAt1g71410 IN007461 107.42 99.81 96.75 97.89 HIO2080 C At1g73660 IN086720127.93 86.29 117.27 HIO2080 C At1g73660 IN086720 127.93 86.29 117.27HIO2080 C At1g73660 IN068250 109.31 92.95 104.37 109.68 HIO2091 DAt1g09870 IN086447 139.97 124.86 65.1 HIO2091 D At1g09870 IN037425100.88 92.75 101.56 98.12 HIO2097 A At5g43250 IN086712 123.39 107.4798.3 HIO2097 A At5g43250 IN086698 119.63 94.3 103.68 203.11 HIO2105 AAt3g07220 IN005625 109.85 91.2 154.61 101.54

TABLE 2 3. Nucleic Acid 4. Polypeptide 5. Putative biochemical 6.Conserved 1. Locus 2. Tair ID seq. GI# seq. GI# function/protein nameprotein domain HIO2019 A At1g17920 gi|42562137 gi|42562138homeobox-leucine zipper family IPR002913 Lipid-bindingprotein/lipid-binding START START domain-containing protein IPR001356Homeobox IPR000047 Helix-turn- helix motif, lambda-like repressorHIO2046 A At5g59010 gi|30697158 gi|22327962 protein kinase-relatedIPR000719 Protein kinase domain IPR002290 Serine/threonine proteinkinase HIO2079 A At1g71410 gi|30698795 gi|15217467 protein kinase familyprotein IPR000719 Protein kinase domain IPR002290 Serine/threonineprotein kinase IPR000357 HEAT HIO2091 D At1g09870 gi|42561877gi|18391081 histidine acid phosphatase family IPR000560 Histidineprotein acid phosphatase HIO2097 A At5g43250 gi|18422310 gi|15239815transcription factor, putative IPR003958 Transcription factorCBF/NF-Y/archaeal histone IPR007124 Histone- fold/TFIID-TAF/NF-YIPR007125 Histone core HIO2080 C At1g73660 gi|30698956 gi|15219517protein kinase family protein IPR000719 Protein kinase domain IPR002290Serine/threonine protein kinase HIO2105 A At3g07220 gi|42563574gi|15231425 transcriptional activator, putative IPR000253 Forkhead-[Arabidopsis thaliana] associated domain

TABLE 3 5. Orthologs 3. Nucleic Acid 4. Poly- peptide Nucleic AcidPolypeptide 1. Locus 2. Tair ID seq. GI# seq. GI# GI# GI# SpeciesHIO2019 A At1g17920 gi|42562137 gi|42562138 gi|42563198 gi|15219456Arabidopsis thaliana gi|51872286 gi|51872287 Gossypium hirsutumgi|51091189 gi|51091201 Oryza sativa (japonica cultivar-group) HIO2046 AAt5g59010 gi|30697158 gi|22327962 gi|30693983 gi|15232406 Arabidopsisthaliana gi|30678717 gi|22328189 Arabidopsis thaliana gi|42568239gi|15237604 Arabidopsis thaliana HIO2079 A At1g71410 gi|30698795gi|15217467 gi|42562252 gi|42562253 Arabidopsis thaliana gi|34912663gi|34912664 Oryza sativa (japonica cultivar-group) gi|13365563gi|54290405 Oryza sativa (japonica cultivar-group) HIO2091 D At1g09870gi|42561877 gi|18391081 gi|50919436 gi|50919437 Oryza sativa (japonicacultivar-group) gi|55730597 gi|55730598 Pongo pygmaeus GI: 19923760 GI:19923761 Homo sapiens HIO2097 A At5g43250 gi|18422310 gi|15239815gi|34906253 gi|34906254 Oryza sativa (japonica cultivar-group)gi|55770762 gi|55770763 Oryza sativa (japonica cultivar-group)gi|21313423 gi|21313424 Mus musculus HIO2080 C At1g73660 gi|30698956gi|15219517 gi|32527768 gi|32527769 Brassica juncea gi|30685720gi|22329643 Arabidopsis thaliana gi|42415350 gi|51535180 Oryza sativa(japonica cultivar-group) HIO2105 A At3g07220 gi|42563574 gi|15231425GI: 50947858 gi|50947859 Oryza sativa (japonica cultivar-group) GI:51965035 gi|51965036 Oryza sativa (japonica cultivar-group) GI: 18397890gi|15231433 Arabidopsis thaliana

Example 2 Analysis of the Arabidopsis HIO Sequence

Sequence analyses were performed with BLAST (Altschul et al., 1990, J.Mol. Biol. 215:403-410), PFAM (Bateman et al., 1999, Nucleic Acids Res.27:260-262), INTERPRO (Mulder et al. 2003 Nucleic Acids Res. 31,315-318), PSORT (Nakai K, and Horton P, 1999, Trends Biochem. Sci.24:34-6), and/or CLUSTAL (Thompson J D et al., 1994, Nucleic Acids Res.22:4673-4680).

Example 3 Recapitulation Experiments

To test whether over-expression of the genes in Tables 1 and 2 alter theseed composition phenotype, protein, digestible protein, oil and fibercontent in seeds from transgenic plants expressing these genes wascompared with protein, digestible protein, oil and fiber content inseeds from non-transgenic control plants. To do this, the genes werecloned into plant transformation vectors behind the strong constitutiveCsVMV promoter and the seed specific PRU promoter. These constructs weretransformed into Arabidopsis plants using the floral dip method. Theplant transformation vector contains a gene, which provides resistanceto a toxic compound, and serves as a selectable marker. Seed from thetransformed plants were plated on agar medium containing the toxiccompound. After 7 days, transgenic plants were identified as healthygreen plants and transplanted to soil. Non-transgenic control plantswere germinated on agar medium, allowed to grow for 7 days and thentransplanted to soil. Transgenic seedlings and non-transgenic controlplants were transplanted to two inch pots that were placed in randompositions in a 10 inch by 20 inch tray. The plants were grown tomaturity, allowed to self-fertilize and set seed. Seed was harvestedfrom each plant and its oil content estimated by Near Infrared (NIR)Spectroscopy using methods previously described. The effect of eachconstruct on seed composition was examined in at least two experiments.

Table 4 lists constructs tested for causing a significant increase inoil, protein, digestible protein or a significant decrease in fiber wereidentified by a two-way Analysis of Variance (ANOVA) test at a p-value≦0.05. These constructs are listed in Table 4. The ANOVA p-values forProtein, Oil, Digestible Protein and Fiber are listed in columns 4-7,respectively. Those with a significant p-value are listed in bold. TheAverage values for Protein, Oil, Digestible Protein and Fiber are listedin columns 8-11, respectively and were calculated by averaging theaverage values determined for the transgenic plants in each experiment.

TABLE 4 Average ANOVA 10. 6. Digestible Digestible 1. Alias 2. Tair 3.Construct 4. Protein 5. Oil Protein 7. Fiber 8. Protein 9. Oil Protein11. Fiber HIO2019 A At1g17920 CsVMV::At1g17920 0.070 0.001 0.247 0.00997.9% 105.3% 100.8% 97.0% HIO2019 A At1g17920 Pru::At1g17920 0.099 0.7120.475 0.782 102.4% 99.5% 100.7% 99.6% HIO2046 A At5g59010CsVMV::At5g59010 0.596 0.258 0.006 0.051 100.7% 101.4% 102.4% 97.4%HIO2046 A At5g59010 Pru::At5g59010 0.757 0.165 0.061 0.029 99.4% 101.9%101.8% 97.3% HIO2079 A At1g71410 CsVMV::At1g71410 0.297 0.033 0.0830.058 99.0% 104.1% 103.1% 95.5% HIO2079 A At1g71410 Pru::At1g71410 0.8520.194 0.097 0.036 99.8% 102.0% 101.7% 97.1% HIO2080 C At1g73660CsVMV::At1g73660 0.755 0.173 0.080 0.044 100.5% 101.9% 101.8% 97.6%HIO2080 C At1g73660 Pru::At1g73660 0.012 0.166 0.062 0.058 95.9% 103.0%102.6% 97.5% HIO2091 D At1g09870 CsVMV::At1g09870 0.685 0.042 0.0060.582 99.6% 102.8% 101.9% 98.0% HIO2091 D At1g09870 Pru::At1g09870 0.3200.103 0.010 0.002 101.3% 102.0% 102.0% 96.9% HIO2097 A At5g43250CsVMV::At5g43250 0.185 0.099 0.108 0.180 98.1% 102.5% 101.6% 98.2%HIO2097 A At5g43250 Pru::At5g43250 0.140 0.056 0.353 0.993 103.6% 98.8%102.1% 98.2% HIO2105 A At3g07220 CsVMV::At3g07220 0.225 0.348 0.2800.751 101.7% 99.0% 100.8% 99.6% HIO2105 A At3g07220 Pru::At3g07220 0.6550.034 0.029 0.000 100.7% 102.1% 101.5% 96.7%

1. A transgenic plant, comprising a plant transformation vectorcomprising a nucleotide sequence that encodes or is complementary to asequence that encodes an HIO polypeptide comprising the amino acidsequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, or an orthologor paralog thereof, whereby the transgenic plant has has an increasedoil content phenotype or an increased meal quality phenotype, relativeto control plants.
 2. The transgenic plant of claim 1, which is selectedfrom the group consisting of plants of the Brassica species, includingcanola and rapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oilpalm, coconut palm, flax, castor, peanut, wheat, oat, and rice.
 3. Aplant part obtained from the plant according to claim
 1. 4. The plantpart of claim 3, which is a seed.
 5. Meal, feed, or food produced fromthe seed of claim
 4. 6. A method of producing oil comprising growing thetransgenic plant of claim 1 and recovering oil from said plant.
 7. Themethod of claim 6, wherein the oil is recovered from a seed of theplant.
 8. A method of producing increased oil content in a plant, saidmethod comprising: introducing into progenitor cells of the plant aplant transformation vector comprising a nucleotide sequence thatencodes or is complementary to a sequence that encodes a HIO polypeptidecomprising the amino acid sequence as set forth in SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQID NO: 14, or an ortholog or paralog thereof, and growing thetransformed progenitor cells to produce a transgenic plant, wherein saidpolynucleotide sequence is expressed, and said transgenic plant exhibitsan increased oil content phenotype relative to control plants.
 9. Aplant obtained by a method of claim
 8. 10. The plant of claim 9, whichis selected from the group consisting of plants of the Brassica species,including canola and rapeseed, soy, corn, sunflower, cotton, cocoa,safflower, oil palm, coconut palm, flax, castor, peanut, wheat, oat, andrice.
 11. The plant of claim 9, wherein the plant is selected from thegroup consisting of a plant grown from said progenitor cells, a plantthat is the direct progeny of a plant grown from said progenitor cells,and a plant that is the indirect progeny of a plant grown from saidprogenitor cells.
 12. A method of generating a plant having an increasedoil content, comprising identifying a plant that has an allele in itsHIO gene that results in increased oil content compared to plantslacking the allele and generating progeny of said identified plant,wherein the generated progeny inherit the allele and have the high oilphenotype.
 13. The method of claim 12 that employs candidate gene/QTLmethodology.
 14. The method of claim 12 that employs TILLINGmethodology.
 15. A feed, meal, grain, food, or seed comprising apolypeptide encoded by the nucleic acid sequence as set forth in SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, or SEQ ID NO:
 13. 16. A feed, meal, grain, food, or seedcomprising a polypeptide comprising an amino acid sequence as set forthin SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12, or SEQ ID NO: 14, or an ortholog or paralog thereof.17. The transgenic plant of claim 1, wherein an increased meal qualityphenotype comprises an increase in available metabolizable energy inmeal produced from seeds of the transgenic plant, relative to controlplants.
 18. The transgenic plant of claim 17, wherein an increase inavailable metabolizable energy comprises an altered protein and/or fibercontent in the seeds of the transgenic plant.
 19. The transgenic plantof claim 18, wherein the protein content is increased and/or the fibercontent is decreased.
 20. The transgenic plant of claim 17, wherein anincrease in available metabolizable energy comprises a decreased fibercontent in the seeds of the transgenic plant.
 21. A method of producingmeal, comprising growing the transgenic plant of claim 1, and recoveringmeal from the plant, thereby producing meal.
 22. The method of claim 21,wherein the meal is produced from seeds of the plant.
 23. A method ofproducing an improved meal quality phenotype in a plant, said methodcomprising: a) introducing into progenitor cells of the plant a planttransformation vector comprising a nucleotide sequence that encodes oris complementary to a sequence that encodes an HIO polypeptidecomprising the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ IDNO: 14, or an ortholog or paralog thereof, and b) growing thetransformed progenitor cells to produce a transgenic plant, wherein thenucleotide sequence is expressed, and the transgenic plant exhibits anincreased meal quality phenotype relative to control plants, therebyproducing the improved meal quality phenotype in the plant.
 24. A plantobtained by a method of claim
 23. 25. The plant of claim 24, which isselected from the group consisting of plants of the Brassica species,including canola and rapeseed, soy, corn, sunflower, cotton, cocoa,safflower, oil palm, coconut palm, flax, castor, peanut, wheat, oat, andrice.
 26. The plant of claim 24, wherein the plant is selected from thegroup consisting of a plant grown from said progenitor cells, a plantthat is the direct progeny of a plant grown from said progenitor cells,and a plant that is the indirect progeny of a plant grown from saidprogenitor cells.
 27. A method of generating a plant having an improvedmeal quality phenotype comprising identifying a plant that has an allelein its HIO gene that results in increased meal quality, compared toplants lacking the allele, and generating progeny of said identifiedplant, wherein the generated progeny inherit the allele and have theincreased meal quality, thereby generating a plant having an improvedmeal quality phenotype.
 28. The method of claim 27 that employscandidate gene/QTL methodology.
 29. The method of claim 27 that employsTILLING methodology.